Project Summary The goal of this STTR grant application is to engineer powerful NK cell-based cytolytic effector cells for therapeutic applications. The cells will combine the selectivity and specificity of T cells and the ability of NK cells to mediate effective cytolytic activity against a wide spectrum of aberrant cells, including those with impaired adhesion properties. To achieve this goal we intend to endow NK92 cytolytic cells with either the entire T-cell receptor (TCR)-CD3 complex or a chimeric antigen receptor (CAR) with the same specificity. In our previous work we developed in vitro evolution approaches to increase the affinities of native TCRs. Using this approach, we have developed high-affinity TCRs and CARs that are specific for different viral- or cancer- associated epitopes. While both of these receptors bind the antigen with high affinity, receptor-mediated signaling will differ due to differences in the receptor's intracellular signaling domain. The kinetics of signaling mediated by two distinct signaling domains of these receptors is expected to affect the efficiency, and possibly the sensitivity, of target cell lysis by NK92-TCR-CD3 and NK92-CAR cytolytic effectors. Because the gene- modified NK92 cells have to be irradiated prior to their transfer to patients, they will be capable of exercising cytolytic activity against target cells for up to 5 days. Thus, the kinetics of target cell destruction becomes a critical factor determining the efficiency of NK92-based cytolytic effectors. The results obtained will enable us to choose the NK92 effector that will exercise the most efficient cytolytic activity against virus-infected and cancer cells in clinical trials. As opposed to adoptively transferred peripheral T cells with either a TCR or a CAR, the potential toxicity of the irradiated NK92-CAR and NK92-TCR-CD3 effectors will be minimized due to the limited persistence of the irradiated effectors. However, NK92 effectors could be transferred to a patient multiple times to generate effective activity against the virus-infected or cancer cells. The novel cytolytic effectors will be available in large amounts and could be utilized for treatment of patients with different genetic backgrounds, thus providing significant advantage over existing T cell adoptive transfer therapies.